In the genome of Arabidopsis thaliana, two genes were identified encoding isoenzymes for C4-hydroxylation of long chain bases (LCB) in plant sphingolipids. Both predicted proteins consist of 258 amino acid residues (77% identity) which show sequence similarity to di-iron-binding enzymes, such as Sur2p and Erg3p from yeast, involved in oxygen-dependent lipid modifications. Heterologous expression of these genes in a yeast sur2Delta-null mutant lacking C4-LCB hydroxylation resulted in the formation of D-ribo-C(18)- and -C(20)-phytosphinganine. The identity and stereochemical configuration of the isolated trihydroxybases was confirmed by electrospray ionization-mass spectroscopy, gas-liquid chromatography-mass spectrometry and 1H-nuclear magnetic resonance spectroscopy. These results represent the first functional identification of SUR2 genes from plants as well as from any organism other than yeast.